THE STRUCTURE OF RIBONUCLEASEshould obtain a mixture of fifteen peptides by complete trypsin digestion of oxidized ribonuclease, which contains 10 lysine and 4 arginine residues. This

THE STRUCTURE OF RIBONUCLEASE

II. THE PREPARATION, SEPARATION, AND RELATIVE ALIGNMENT

OF LARGE ENZYMATICALLY PRODUCED FRAGMENTS

BY ROBERT R. REDFIELD AND CHRISTIAN B. ANFINSEN

WITH THE TECHNICAL ASSISTANCE OF JUANITA COOKE

(Prom the Laboratory of Cellular Physiology and Metabolism, National Heart Institute, National Institutes of Health, Department of Health, Education,

and Welfare, Bethesda, Maryland)

(Received for publication, November 25, 1955)

Ribonuclease consists of a single, cross-linked chain (1) containing ap- proximately 124 amino acid residues (2, 3). The N-terminal sequence of this chain, together with certain deductions concerning its gross structure, has been presented earlier (1). In the present paper we wish to report on the results of one approach to the elucidation of the complete sequence of this protein.

Sanger concluded in his review in 1952 (4) that “Probably the chief need in this field is for techniques for the specific breakdown of proteins into larger peptides and for the fractionation of such peptides.” Of the proteolytic enzymes available, trypsin appears to have the sharpest specificity since no evidence for peptide bond cleavage other than that expected from the fundamental studies of Bergmann and Fruton (5, 6) has been demonstrated in protein degradation studies on insulin (7) or corticotropin (S-10). Since trypsin catalyzes the hydrolysis of peptide bonds involving the car- boxy1 groups of the basic amino acid residues, lysine and arginine, one should obtain a mixture of fifteen peptides by complete trypsin digestion of oxidized ribonuclease, which contains 10 lysine and 4 arginine residues. This has been the basis of the approach to the structure of ribonuclease selected by Hirs, Moore, and Stein (3), who have been able to isolate and characterize thirteen peptides of distinctly different composition from ribonuclease which account for all the amino acid residues of the molecule. Eleven of these peptides contain 1 basic amino acid residue. Two of the peptides, one of which is from the N-terminal sequence of ribonuclease, contain 2 basic residues.

Our own approach has been to mask the lysine residues by dinitrophenylation of their c-amino groups, rendering them unsusceptible to trypsin digestion, and, theoretically, allowing cleavage at the arginine residues only. There should be produced, under these circumstances, five rather large peptides from ribonuclease having an average size comparable to the

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386 STRUCTURE OF RIBONUCLEASE. II

peptide chains of insulin. Each of these peptides, then, should lend itself more readily to sequence studies than the much larger, intact protein. Furthermore, by the isolation from other types of digests of peptides involving arginine, and by determination of these arginine-containing sequences, the relative alignment of the large trypsin-produced peptides would be permitted. Such studies on the production, separation, charac- terization, and relative alignment of trypsin-produced fragments of oxidized, dinitrophenylated ribonuclease are reported below, together with ancillary data which permit the partial reconstruction of the amino acid sequence of the molecule.

Materials

Ribonuclease-As in previous studies, the present work was carried out on a single lot of commercial ribonuclease (Armour, Lot 381059), some of the chemical and physical properties of which have been described earlier (1). This preparation gave essentially a single peak on IRC-50 chromatography (ll), indicating a very low initial level of the component designated as component “B.”

The “B” component (or a material chromatographically similar to it) appears to be formed during storage at low temperature (Hirs, personal communication), and our working material has undoubtedly become en- riched with it. The assumption has been made, however, that the chemical changes taking place during the conversion from “A” to “B” do not influence the sequence of amino acids in the molecule, but are concerned with functional groups involved in net charge and charge distribution (e.g. amide groups). Indeed, recent titration and spectrophotometric studies by Tanford and Hauenstein (12) indicate that the two chromatographic species differ by only a single free carboxyl group.

Performic acid oxidation of ribonuclease was performed observing the precautions outlined by Thompson (13) and by Hirs (14) to avoid the chlorination of tyrosine .

The oxidized ribonuclease was dinitrophenylated according to the basic procedure of Sanger (15).

Trypsin-The material used was commercial Armour, three times crys- tallized, salt-free trypsin (Lot M68009). In order to lower, preferentially, the relative activity of any chymotrypsin contaminant, the trypsin was initially subjected to an incubation in ~/16 hydrochloric acid for 24 hours at 37” at a concentration of 1 per cent, as suggested by Northrop and Ku- nitz (16).

Results

Kinetics of Trypsin Digestion-A 1 per cent suspension of dinitrofluorobenzene-treated, oxidized ribonuclease in phosphate buffer, pH 7.9, ionic


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R. R. REDFIELD AND C. B. ANFINSEN 387

strength 0.16, was made 0.020 per cent with respect to acid-treated trypsin and incubated at 37”. The undigested ribonuclease derivative is rendered completely soluble after about 10 minutes of digestion. Aliquots containing 10 mg. of the substrate were removed (Fig. 1) at various times, 0.1 cc. of 0.2 M dinitrofluorobenzene was added immediately, and the aliquots were dinitrophenylated according to Levy (17). After extracting the excess reagent, the samples were dried in vacua and hydrolyzed for 18 hours with 6 N HCl in sealed tubes at 105”. The hydrolysates were diluted, each divided into two equal parts, extracted eight to ten times with half volumes of peroxide-free ether, and concentrated to dryness by evaporation at 60”. The ether-soluble fraction was then chromatographed according to Levy (17). The complex zones containing DNP-aspartic and DNP-glutamic acids were rechromatographed by using the tert-amyl alcohol-phthalate buffer (pH 5) system of Blackburn and Lowther (18). All separated DNP- amino acids were eluted and estimated spectrophotometrically at 360 rnp.

It was found in preliminary experiments that DNP-cysteic acid was not adsorbed by t.he acid talc-kieselguhr columns generally used for desalting solutions of water-soluble DNP-amino acids (19). The aqueous residues, after ether extraction, were therefore evaporated in vacua to a dry film and DNP derivatives were dissolved away from the bulk of the salt by small washes of ethanol-O.1 N HCl (4: 1). This fraction was chromatographed in two dimensions (17) and determined as above.

The absence of DNP-glycine or DNP-proline as N-terminal groups was demonstrated by repeating the above procedures on hydrolysates carried out in concentrated HCl, in which a portion of these otherwise highly labile derivatives survives hydrolysis (20). Correction factors for loss during hydrolysis (20)’ and chromatography (determined independently by subjecting synthetic derivatives to the same procedures)2 were applied, and the results were expressed as moles of DNP-amino acid per mole of bis- DNP-lysine, the N-terminal residue of ribonuclease.

The results of the kinetic study of digestion with acid-treated trypsin are shown in Fig. 1. Two glutamic acid terminal peptides, one aspartic acid terminal peptide, and one cysteic acid terminal peptide are formed in addition to the peptide containing the N-terminal bis-DNP-lysine residue already present in DNP-oxidized ribonuclease. Of the new end-groups formed, DNP-cysteic acid appears most slowly. As shown, the yield of this end-group was 0.75 mole per mole of ribonuclease at 8 hours and about 0.85 mole per mole at 24 hours. The use of trypsin which has been treated to inactivate other contaminating proteolytic enzymes is of distinct advan- tage. Thus it has been found in kinetic experiments similar to those sum-

1 Recovery of DNP-cysteic acid after hydrolysis, 95 per cent. 2 Chromatographic recovery, DNP-aspartic and DNP-glutamic acids, 90 per cent;

bis-DNP-lysine, 80 per cent; DNP-cysteic acid, 90 per cent.


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marieed in Fig. 1 that commercial trypsin, used at the same level of tryptic activity, led to the appearance of as much as 0.4 mole per mole of other non-specific end-groups (Ser, Thr, Ala, Val) as compared to 0.2 mole per mole (Fig. 1) after 8 hours of digestion.

In the preparation of digests for fractionation studies, a digestion time of 8 hours with acid-treated trypsin was selected. Under these conditions, non-tryptic hydrolysis was minimal and specific cleavages were essentially complete (Fig. 1).

Fractionation of Digestion Mixture

The digestion mixture was adjusted to pH 2.5 to 3.0 by the addition of hydrochloric acid, yielding an acid-soluble and an acid-insoluble fraction.

l GLU A SER 8 VAL + CYSO,H tj y 2.5 0 ASP A ALA 0 THR

p Fe.0 3;’

&g 1.5 ’

; 2 1 1.0

z c 0.5

dd ==f 0

0 4 8 12 16 20 24 DIGESTION TIME (HOURS)

FIG. 1. Production of N-terminal amino acid groups during digestion of DNP- oxidized ribonuclease by acid-treated trypsin. See the text for digestion conditions.

The precipitate, containing the bulk of the material, was washed three times with 0.02 M acetic acid, and the washings were combined with the supernatant fraction.

Fractionation of Acid-Soluble Fraction

By end-group analysis the only important constituent of this fraction was found to be the aspartic acid terminal peptide. Much smaller amounts of a variety of other peptides were present, including most of the materials formed by non-tryptic hydrolysis.

The aspartic acid terminal peptide was readily fractionated from these contaminants by simple electrophoresis on paper or a cellulose powder block after the material had been desalted by adsorption on a talc-kieselguhr column (19). The migration of this component is as indicated in Fig. 2 (Band I).


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Fractionation of Acid-Insoluble Fraction

Electrophoresis-The separation of the constituents of this fraction was not possible by the usual electrophoresis procedures with dilute aqueous buffers, owing to the pronounced adsorption of these compounds on the

CMS. FROM ORIGIN

RF o.oo-

""'"'"4 0 0.25 0 @@O

O@O

0.50 A d

0 .

0.75’

FIG. 2. Fractionation of the acid-insoluble fraction. Electrophoresis was done on Whatman No. 3 paper wet with 8 M urea-O.04 M collidine-acetate buffer, pH 7.9,

and run for 12 hours at 800 volts, 12 ma., under toluene (21). Descending chromatography was done on Whatman No. 3 paper by using the system: set-butanol 60, tert- butanol20, 2,4,6-collidine 20, ammonium hydroxide 2, glacial acetic acid 0.25, water

100 (volume per volume). The papers were allowed to equilibrate for 2 hours in an atmosphere of the aqueous phase before introduction of the organic phase. Desig-

nation of peptides, Band I is the N-terminal aspartic acid Peptide C, present in

trace amounts in the acid-insoluble fraction; Band II, RF 0.27, is the N-terminal glutamic acid Peptide B; Band III, present in trace amounts, was not investigated; Band IV, RF 0.54, is the N-terminal lysine Peptide A; Band V, RF 0.46, is the N-

terminal glutamic acid Peptide E; Band VI, RF 0.22, represents unhydrolyzed Pep- tides C + D; Band VII, RF 0.15, is the N-terminal cysteic acid Peptide D.

several types of supports tried. However, by wetting the paper (What- man No. 3) with buffer at a pH above 7, made 8 M with respect to urea, adsorption became negligible and the components migrated in rather sharp bands, particularly when the high voltage gradient technique of Michl (21) was used. In this way, the lysine terminal peptide (Band IV) and one glutamic acid terminal peptide (Band II) were readily separated (Fig. 2).


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The charge densities of the other components were sufficiently similar to prevent clean separation at this pH. At lower pH values, the components either became insoluble or were progressively more strongly adsorbed. The choice of pH range was further restricted, since high pH values had to be avoided because of the lability of the DNP derivatives in alkaline solution.

Counter-Current Distribzction-Fractionation of the trypsin products could also be accomplished by counter-current distribution. The most useful solvent systems were mixtures of set-butanol, tert-butanol, collidine, and buffers at a pH of 7 or above. The fractionation could be followed by absorption measurements at 360 mp of the yellow DNP derivatives. A fractionation involving 150 transfers by a system composed of sec-butanol- tert-butanol-0.025 M phosphate buffer, pH 6.80 (75:25: loo), allowed the separation of the lysine terminal peptide, two distinct peaks containing glutamic acid as the major end-group and one slow peak containing aspartic and cysteic acids as the end-groups. A complex mixture of peptides, containing about 20 per cent of the total sample, remained in the first few tubes. Except for a portion of the N-terminal lysine component, none of these fractions was found, by end-group analysis, to be of sufficient purity for analysis without preliminary electrophoretic separation.

The pattern of fractionation was very similar when distribution chromatography was employed with either cellulose columns or paper sheets. In view of the simplicity afforded by the use of a cellulose support, distribution was subsequently carried out on paper. Although streaking was no serious problem with these systems, the use of such a support suffered from the retention of variable amounts of the peptides at the point of applica- tion. This was largely avoided by using a minimal volume of solvent (1 per cent trimethylamine) to dissolve the sample so as to make repeated ap- plications unnecessary and by inclusion of collidine in the developing solvent. A diagram summarizing the chromatographic purification of the electrophoretic components is shown in Fig. 2.

A combination of the electrophoretic and distributive chromatographic techniques permitted the clean fractionation of all five peptides present in the digestion mixture.3 Elution of the compounds from paper was best

3 Numerous other methods were examined for their possible value in fractionation.

Owing to the large size and high level of both non-polar and polar groups present in the peptides under investigation, techniques involving resin columns, anionic cellulose columns, and most paper chromatographic procedures were unusable, on account of strong adsorption effects. A gradient salting in procedure, in which high ethanol (33

to 66 per cent) concentrations, low ionic strengths (0.002 to 0.005)) and a continuously changing pH (3 to 9) were employed, yielded very encouraging enrichments, up to 80

per cent, for single constituents, as reported earlier (22). The salting in solution was allowed to flow over a column of very fine glass beads upon which was precipitated the digest mixture in a highly dispersed state, after the basic procedure of Zahn and Stahl


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effected by the use of an 8 M urea solution. After adsorption of the colored derivatives in the eluates on short talc-kieselguhr columns, the columns were washed with water, and the DNP peptides were eluted rapidly with ethanol-l per cent trimethylamine (2:l) and concentrated to dryness in vacua. The dry materials were stored at 0”.

Analysis of Purijled Peptides-After hydrolysis with 6 N HCl under the conditions listed in Table I, the hydrolysate (or the ether-insoluble fraction of the hydrolysate in the case of a DNP peptide) was taken to dryness in vucuo over KOH pellets. The residue was taken up in 3 cc. of water and dinitrophenylated and chromatographed according to Levy (17):’ 6

The peptides as isolated in the previous section (Fig. 2) were found to be homogeneous by end-group analysis. Peptides A and C in Table I were analyzed by using the same DNP peptide (dinitrophenylated according to Levy (17)) for both the end-group anaylsis and amino acid residue analysis. Duplicate samples of Peptides B, D, and E were analyzed for their end- groups and amino acid content separately.

The data in Table I (Peptide C) show that the destructive hydrolysis of the N-terminal DNP-aspartic acid is reflected in an increase of free aspartic

(23). Further investigations demonstrated, however, that this method did not per-

mit complete purification of any single peptide and was therefore abandoned. 4 The occurrence of certain unidentified artifact spots in the Levy two-dimensional

chromatogram encountered in these studies deserves comment. One artifact run- ning just slower than phenylalanine in the “toluene” direction and just slower than leucine in the “phosphate” direction must be distinguished from spots due to these

amino acids themselves. An orange-colored artifact may occur in the glycine area, but is usually slightly faster than glycine in the “toluene” direction. Another orange-colored artifact may occur in the valine area, but is usually sufficiently slower in the “toluene” direction to allow separation. Two other artifact spots appear to be associated with decomposition products of t-DNP-lysine or dinitrophenylation of

these products. These occur in the neighborhood of bis-DNP-lysine and have a mo- bility in the “phosphate” direction equal to that of bis-DNP-lysine, but have different mobilities in the “toluene” direction. One of these is orange and has an RF in “toluene” just slower than that in valine. Another is yellow and has an Rp in the “toluene” solvent between bis-DNP-histidine and DNP-leucine. Care must be

taken not to confuse these spots with that due to bis-DNP-histidine, which moves slightly faster than the artifact spots in the “phosphate” direction.

5 Two areas of the two-dimensional chromatograms frequently required rechroma-

tography in the tert-amyl alcohol-phthalate buffer (pH 5) system of Blackburn and Lowther (18), with papers soaked in dilute buffer (0.05 M) to allow sufficient migration of DNP-aspartic acid and DNP-glutamic acid. One of these is the DNP-dicarboxylic acid area as noted above. The other is that area occupied by DNP-serine, DNP-methionine sulfone, and DNP-threonine, when DNP-methionine sulfone is a constituent. In these cases, a total density figure for the complex is determined, and

the per cent contribution due to each of the components is determined after rechroma- tography. This fraction of the total density is then taken as the analytical value rather than the absolute figure as determined by the second chromatogram.


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TABLE I Amino Acid Analysis of Peptides Produced by Trupsin

Peptides A and C were analyzed after dinitrophenylation. Peptides B, D, and E were analyzed as the free peptides. F = Levy factor (17) except as described in the text.

Lysine terminal Peptide A*

G

Bis-DNP- Lys.. ..o .9

DNP-Asp ... Asp ....... Glu.. ...... 2.1 f O.( Ser.. ...... .O.l Thr.. ..... 1.0 f 0.: Met02 ...... Gly.. ...... 0.2 f 0.: Ala ........ 3.0 f 0.: Pro. ........ Val ........ Leu ....... Ileu ....... Phe ....... 1.1 f O.( His ........ Lys.. ..... .0.7 ZIZ 0.: Tyr ....... CySOaH**. Arg**. ..... 1.1 f 0.f

Total. ...

lutamic acid terminal

Peptide Bt

3.1 2.0 7.5 0.8 3.2 0.1 2.1

3 2 7-8 1 3

1.0 1.2 1.0 0.9 0.9

13-24

-

IP mpartic acid terminal

Peptide Ct:

8 : a ; ge

.$ 5

0.7 1.4

1.0

1.0

0.8

1.0

6 434! 3640

-

(

-

Jysteic acid tennina Peptide DO

5.9f0.5 6 4.3fO.l 4 4.7 5 3.6 4 0.9fO.l 1 1.2f0.2 1 3.2fO.l 3 1.3fO.l 1 4.6 5

\.: ; i

0:5f0.2 17 1.9f0.5 2-3’ 1.6kO.2 27 4.4f0.5 4-5 l.oZto.o 1

- 1

t Glutamic acid

.erminal Peptide El/

4.1&O. 3.1&O. 3.4&O. 3.3&O.

2.1&O. 3.7&O. 2.3~0. 3.9&O.

1.7*0.

‘1.0 1.1&O. 1.8&O. 2.4&O. 1.6~0.

-

-

4 3 34 3

2 4 2-3 4

i 2

1 27 2-3lj 2-37 2

* Peptide A, average of two analyses performed on separate 6 N HCl hydrolysates of the DNP peptide (18 hours at 105”). The average unit residue figure, D360 X F, based on the average value for 1 residue, was 0.217.

t Peptide B, single analysis of a 6 N HCl hydrolysate of the free peptide (18 hours at 105”). The unit residue figure, D 36~ X F, based on the average value for 1 residue, was 0.218.

$ Peptide C, single analysis of a 6 N HCl hydrolysate of the DNP peptide (18 hours at 105”). The unit residue figure, Da60 X F, based on the average leucine and threonine value, was 0.796. Absolute identification of leucine was made by chromatography of an aliquot of the hydrolysate with n-butanol-water (85:15) (24).

8 Peptide D, average of two analyses performed on separate 6 N HCl hydrolysates of the free peptide (22 and 70 hours at llO”), with the average deviation of the mean indicated. The average unit residue figure, 0360 X F, based on phenylalanine = 1


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TABLE I-Concluded residue per mole, was 0.118. Only the values obtained on the shorter hydrolysis have been taken for the notably labile residues (serine and threonine) and only those for the longer times for the amino acids which increased with longer hydrolysis (leucine-isoleucine and valine).

11 Peptide E, average of four analyses performed on separate 6 N HCl hydrolysates of the free peptide (18 and 69 hours at 105” and 22 and 70 hours at llO”), with the average deviation of the mean indicated. The average unit residue figure, DM.O X F, based on phenylalanine = 1 residue per mole, was 0.065. Only the values obtained on the two shorter hydrolyses have been taken for the notably labile residues (serine and threonine) and only those for the longer times for the amino acids which increased with longer hydrolysis (leucine-isoleucine and valine).

7 The values for these amino acids, present as their mono-DNP derivatives in the peptide, have given erratic low values. See the text for assignment of assumed residues.

** These values have been corrected for the 10 per cent chromatographic loss of water-soluble DNP derivatives routinely observed.

acid in the hydrolysis mixture. The sum of these aspartic acid residues gives a figure of 2.1 residues of aspartic acid in Peptide C. The value for lysine, present as e-DNP-lysine in the peptide, gave a low value of 0.8 mole per mole when the Levy factor, 0.64, was applied. Low values were encountered in most other instances involving r-DNP-lysine, and, therefore, a factor of 0.8, which would lead to a residue value of 1.0 for Peptide C, was taken in subsequent analyses.

The N-terminal lysine Peptide A is clearly a 10 residue peptide. The value for lysine is low, despite the use of the higher analysis factor as noted above, indicating an unusual lability of c-DNP-lysine in this peptide.

The analysis for the N-terminal glutamic acid Peptide B indicates the presence of 23 to 24 amino acid residues. The choice between 7 or 8 residues for serine cannot be made on the basis of these data. The value for histidine has been based on the use of the factor for histidine as given by Levy (17), which measures histidine as ar-DNP-histidine. The imidazole group is difficult to dinitrophenylate in free histidine (17), in contrast to histidine in peptidic linkage. The histidine in these peptides is in the form of imidazole-DNP-histidine. The extent of dinitrophenylation of the free a-amino group to give bis-DNP-histidine under the Levy conditions has not been investiga.ted. Therefore, the use of this factor must be taken as a first approximation and is of value only in comparing the figures obtained for the various histidine-containing peptides.

The N-terminal cysteic acid Peptide D appears to contain 43 to 45 residues. Rigorous analysis of peptides of this size would require several sets of analyses, with extrapolation to zero time hydrolysis, as used by Smith and Stockell (25). The approach to structure selected here does not, however, require preliminary analyses of absolute precision, since the objective


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of this approach has been to degrade the largest units (the protein as a first step) into progressively smaller specific subunits. Analyses of the smaller component peptides in which the analyses become unequivocal should later resolve the uncertainty of the analysis of the large unit.

The approximate amino acid composition of .the N-terminal glutamic acid Peptide E indicates that this peptide does not contain arginine and must have occupied a position at the C-terminal end of the ribonuclease molecule. Because of its size (36 to 40 residues), the exact residue number for certain amino acids is again uncertain. The amino acids histidine, lysine, and tyrosine have given the most erratic values. These amino acids, occurring as their mono-DNP derivatives in the peptides isolated, are apparently more labile than other unsubstituted amino acid residues. Since low values for lysine were noted in the case of Peptide A, it is clear that the analyses for lysine in Peptides D and E can indicate either 2 or 3 residues. The average value of 0.5 for histidine in Peptide D, corresponding to 1 residue, suggests the assignment of 2 residues of histidine to Peptide E. These 3 histidine residues, together with the 1 residue of histidine in Peptide B, account for the 4 residues determined in the molecule by Hirs et al. (2).

Preliminary end-group and quantitative amino acid analysis of the constituent from Band VI, RF 0.39 (Fig. 2), suggests that this component represents the unhydrolyeed sequence, Peptides D + E. The component from Band VI, RF 0.22, has an aspartic acid N-terminal residue and probably represents the unhydrolyzed sequence, Peptides C + D.

Sequences Containing Arginine-As indicated by the kinetics of the trypsin hydrolyses discussed above, five fragments are produced during tl_e digestion of DNP-oxidized ribonuclease in agreement with the known speci- ficities of trypsin. One of these, the N-terminal fragment, may be detected by its content of bis-DNP-lysine. A second fragment, derived from the C-terminal end of ribonuclease, is characterized by its lack of arginine. To enable the positioning of t’hese two peptides with respect to the remaining three requires the determination of the amino acid sequences involving 3 of the 4 arginine residues.

Acid Hydrolysis-As one approach to this problem, partial acid hydrolysates of native ribonuclease were prepared by incubation, at 37”, in 11.5 N HCl for 48 hours. After removal of HCl by repeated evaporation in vacua the total digest was treated with DNFB according to Sanger (15). Excess reagent, dinitrophenol, dinitroaniline, and dinitrophenylated amino acids or small peptides not containing arginine were removed by ether extraction from the alkaline solution and after acidification. The DNP peptides insoluble in ether were then extracted into n-butanol and the solvent was removed in vacua as rapidly as possible to avoid esterification. The


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resulting mixture was streaked on Whatman No. 3 filter paper and subjected to ionophoresis at pH 3.7 in pyridine-acetic acid buffer according to Michl (21). At this pH the free carboxyl groups of aspartic and glutamic acids are only partially ionized and consequently those DNP peptides containing arginine should move towards the negative electrode. As shown in Fig. 3, four main bands were obtained along with several minor bands which were not eluted. Bands A, B, C, and D were eluted with water,

B I,,-C-C-ORIGIN-

FIG. 3. Isolation of arginine-containing peptides from partial acid hydrolysates

of ribonuclease. A, electrophoresis (21); B, subsequent filter paper chromatography (set-butanol-3 per cent NH3) of electrophoretic bands.

c3

0 0

streaked on Whatman No. 3 filter paper, and chromatographed in the descending direction with see-butanol-3 per cent NHI. As shown in Fig. 3, each of the ionophoretic bands divided into several components. The major components were eluted with 6 N HCl and hydrolyzed overnight in sealed tubes at 105”. The terminal amino acid of each component (except Component B, which was not studied) was determined by two-dimensional chromatography of the ether-soluble material according to Levy (17). These chromatograms resulted in unequivocal identification and showed in each case only single end-groups. The aqueous portion of each hydrolysate


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wa.s evaporated in vacua to remove HCl and the qualitative amino acid composition was determined by two-dimensional chromatography with 80 per cent phenol-l per cent ammonia-coal gas in one dimension and butanol- acetic acid-water (4 : 1: 5) in the second.

Fig. 4 summarizes the results of these experiments. As shown, peptides of increasing basicity were found closest to the negative electrode of the ionophoresis strips.

The acid hydrolysis studies account for two of the four arginine sequences of ribonuclease, as discussed in the section below on the reconstruction of the ribonuclease chain. The remaining two sequences were undoubtedly carried down in the large precipitate which remained insoluble in butanol following dinitrophenylation of the partial acid hydrolysates. Examina- tion of the amino acid composition of the other chromatographic species

0 COMPOSITION PROBABLE

ORIGIN SEQUENCE

DNP-Asp [ARC, GLU] [-ASP-ARC-GLU]-

[-ARG-GLU]-

ARG, LEU] [-SER-ARG-ASP-LEU]

[-SER-ARC-]

FIG. 4. The N-terminal amino acids and compositions of the arginine-containing

peptides isolated as shown in Fig. 3.

shown in Fig. 3 indicated that they represented further hydrolysis products of the peptides listed in Fig. 4.

Pepsin Digestion-Attempts were made to isolate other arginine-containing sequences from pepsin digests of oxidized ribonuclease. A 1 per cent solution of the oxidized protein in 0.01 N HCI was incubated for 15 hours at 37” in the presence of 0.01 per cent crystalline pepsin. The solution was taken to dryness in vacua. Aliquots were applied to Whatman No. 3 paper and subjected to ionophoresis (4 hours, 1500 volts) at pH 3.7 as described above (21). The pattern of ninhydrin-positive material and the location of Sakaguchi-positive components indicated the presence of four electropositive zones containing arginine. The most electropositive zone, which gave the strongest Sakaguchi test for arginine, was eluted with water, evaporated to dryness to remove pyridine acetate, and reapplied to Whatman No. 3 paper for a second ionophoresis (4 hours, 1500 volts) at pH 6.8 with the same buffer components. Five major subfractions (all migrating towards the negative electrode) were well separated and con-


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tained arginine as indicated by a positive Sakaguchi test. These were eluted and analyzed qualitatively, after hydrolysis, by two-dimensional paper chromatography. These preliminary chromatograms indicated a rather complex composition for all but the least electropositive component. Small aliquots of the latter peptide were rerun at pH 3.7 and at 6.8 and appeared homogeneous under both conditions.

Acid hydrolysis of an aliquot of this peptide, followed by two-dimensional chromatography in the phenol-butanol-acetic acid system as used above, indicated the presence of the following: (Asp, Arg, Glut, Ala, Thr, MetOz, His, Ser%J. The presence of 2 to 3 equivalents of serine and of 2 equivalents of glutamic acid per mole was suggested by the comparison with con- trol chromatograms with differing mole ratios of the various amino acids found in the preliminary analysis of the fragment. The peptide was treated with DNFB according to Sanger (15) and, after extraction with ether at both alkaline and acid pH to remove excess reagent and soluble contaminants, the yellow reaction mixture was taken to dryness. The DNP peptide was removed from the major portion of the salt by extraction of the dry residue with glacial acetic acid, followed by rapid evaporation. Hy- drolysis with HCl, followed by chromatography (17) as described above, yielded DNP-glutamic acid as the only end-group. The aqueous solution after removal of the end-group with ether contained all the amino acids found above.

Samples of the DNFB-treated peptide (1 hmole on the basis of quantitative end-group analysis (17)) prepared as described above were dissolved in 0.5 ml. of phosphate buffer, pH 8.0, ionic strength 0.15, and digested with trypsin, which was present at a level of 0.4 per cent. After incubation at 37” overnight, the solutioh was acidified to about pH 2 and extracted with butanol. The butanol extract was washed twice with small amounts of water and the butanol phase was taken to dryness and hydrolyzed overnight in concentrated HCl at 105”. The end-group was determined by chromatography as above and proved to be DNP-glutamic acid. The aqueous phase contained only arginine. These find- ings indicate that the partial structure of the peptide must be .Glu.Arg.Glu.(Asp,Ala,Thr,MetOz,His,Ser&, since arginine must be followed either by a cysteic acid or by a dicarboxylic amino acid according to the trypsin digestion data described above and since the sequence, .Arg.Asp, has already been shown to occur in another area of the protein (Fig. 4 and Table II).

The experiments above establish the presence in ribonuclease of the following arginine-containing sequences: .Ser(Arg,Asp,Leu), Asp.(Glu,Arg), and Glu.Arg.Glu.(Asp,Met,His,Sers3,Ala,Thr).

Sequence Studies; N-Terminal Aspartic Acid Pepticle C-The amino acid sequence of the N-terminal aspartic acid hexapeptide was determined as


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398 STRUCTURE OF RIRONUCLEASE. II

follows: The peptide was eluted from the electrophoretic strips and dinitrophenylated (17). The material was concentrated to dryness in vacua and subjected to partial acid hydrolysis in 11 N HCl at 37” for 16 hours. After dilution with water, the solution was extracted five times with half volumes of ether, and the combined extracts were evaporated to dryness. The solution was then extracted three times with ethyl acetate, and the extracts were back-washed with water. The combined ethyl acetate ex-

TABLE II

Sequence Data for N-Terminal Aspartic Acid Peptide C

N-Terminal end of Peptide C

1 +;.5 2 +18.0 3 +15.0 4 +11.0

5 +7.5

1+ 2+

1+

2+

DNP-Asp “

0 DNP-Asp Leu DNP-Asp.Leu

Dinitrophenol 0

DNP-Asp 0

“ Leu

C-Terminal end of Peptide C

DNP-Asp-

NHz* DNP-Asp-

NHs.Leu*

1 -4.5 2f DNP-Arg 0 DNP-Arg

2 -1.0 1+ DNP-Thr {

e-DNP-Lys,

ASP, Ax I DNP-Thr-

(Lys,Asp.-

Ard 3 +1.5 If DNP-Asp Arg DNP-Asp.Arg

* Zones 1 and 4 and Zones 2 and 5 had the same composition after complete hydrolyses. The pairs of zones which migrated more slowly towards the anode must,

therefore, have been in their amide forms.

tracts were evaporated to dryness. Both the ether and ethyl acetate extracts were subjected to paper electrophoresis in 0.03 M phosphate buffer, pH 7.0, at 230 volts, 10 ma., for 7 hours. The yellow, electrophoretically separated zones were eluted, desalted on a talc-kieselguhr column as described above, and hydrolyzed for 18 hours at 105” in sealed tubes. The hydrolysates were extracted with ether and chromatographed. The aqueous fractions were concentrated to dryness and chromatographed for free amino acids (26). The results giving the sequence at the N-terminal end of Peptide C are summarized in Table II.

The aqueous portion of the partial acid hydrolysate was concentrated to dryness and dinitrophenylated. The solution was then extracted with


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ether, acidified, and reextracted with ether and ethyl acetate as above. The material remaining in the aqueous solution, representing cr-DNP-arginine and peptides involving arginine, was concentrated to dryness and subjected to paper electrophoresis as above. The yellow electrophoretic zones were eluted, desalted, and hydrolyzed with 6 N HCl at 105” for 18 hours. Each of these hydrolysates was then extracted with ether and analyzed for its N-terminal DNP-amino acids (17). The aqueous solution was analyzed for free amino acids (27). The results, giving the sequence at the C-terminal end of Peptide C, are summarized in Table II.

The sequence of Peptide C can thus be written as AspNHz.Leu.Thr.Lys.- Asp.Arg.

N-Terminal Lysine Peptide A-A 1 per cent solution of Peptide A (ap- proximately 0.5 pmole) in phosphate buffer, pH 7.9, ionic strength 0.16, was digested for 17 hours at 25” in the presence of 0.01 per cent chymotrypsin (1 per cent relative to substrate). The total digest was dinitrophenylated (17). After extraction of the excess reagent and evaporation to dryness, the residue was dissolved and subjected to paper electrophoresis in 0.033 M phosphate buffer, pH 7.01, as above. The two darkest zones were eluted, desalted, and chromatographed on paper with methyl ethyl ketone-propionic acid-water (75:25:30) (27). The eluted zones were hydrolyzed with 6 N HCl for 18 hours at 105” in sealed tubes. After ether extraction of the diluted hydrolysates, the DNP-amino acid end-groups mere identified by paper chromatography (17, 18). The aqueous fractions were evaporated to dryness in vacua, and aliquots were taken for free amino acid chromatography (26) and analysis according to Levy (17). The following peptides were characterized: a peptide migrating +6.3 cm. upon electrophoresis, and chromatographing with an RF 0.77, was shown to be DNP-Glu.Arg; a peptide migrating +17.5 cm. upon electrophoresis, and chromatographing with an RF 0.95, had the amino acid composition bis- DNP-Lys.(Glu,Thr,Alas,c-DNP-Lys).Phe. In the latter peptide, phenylalanine is placed outside the parenthesis at the C-terminal end of this peptide in accordance with the known specificity of chymotrypsin. The N-terminal sequence has been previously determined (1) as Lys.Glu.Thr.- Ala. The sequence of Peptide A, occupying the N-terminal position in ribonuclease, can thus be written as Lys.Glu.Thr.Ala.(Alaz,Lys).Phe.Glu.Arg. A peptide with this same composition can be constructed from data obtained on trypsin hydrolysates of oxidized ribonuclease by Hirs, Stein, and Moore (28).

Other Peptide Fragments

During the course of chymotrypsin digestion studies on the large trypsin- produced peptides described above, and from preliminary experiments on the nature of the disulfide bridges of ribonuclease, a considerable number


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of peptide fragments have been isolated and analyzed. Two of these are of immediate interest in the partial reconstruction of the ribonuclease molecule as presented in a later section of this paper. A peptide liberated by chymotrypsin was isolated by filter paper electrophoresis and paper chromatography of digests of the peptide designated Peptide D. Its quantitative composition -Ser. (Leu or Ileu , Thr , CySO,H, Asp) .Arg. was established by two-dimensional chromatography by the method of Levy for hydrolysates of the electrophoretic zone in question. The serine end-group was determined by the usual procedure (17).

The cysteic acid-containing peptide (CySOsH,Asp,Arg,Glu,Gly,Ser) was isolated as part of a disulfide “bridge” from subtilisin digests of ribonuclease, by electrophoresis according to Michl, and analyzed by two-dimensional chromatography (26) after rupture of the disulfide linkage with performic acid.

As indicated in the reconstruction given in the section below, the amino acid composition of these fragments permits the subdivision of some of the large trypsin-produced fragments into smaller groups of residues.

C-Terminal Sequence-The data permitting the reconstruction of the C-terminal sequence of ribonuclease are summarized below. Digestion with carboxypeptidase had earlier established the presence in ribonuclease of valine as the C-terminal residue (1, 29). Other residues were released during prolonged digestion. In later studies (unpublished data of Anfinsen and Richards), it was found that longer digestions with another more thor- oughly diisopropyl fluorophosphate-treated carboxypeptidase preparation yielded the amino acids serine and alanine in addition to valine, suggesting the presence at the end of the chain of the grouping -(Ala,Ser).Val. Hy- drazinolysis according to Ohno (30) has confirmed the presence of valine as the C-terminal residue. In unpublished experiments by Niu and Fraenkel-Conrat (31), partial hydrazinolysis was found to release, in addition to valine, the peptide -Ser.Val. Finally, limited pepsin digestion of native ribonuclease results in the quantitative liberation of the tetrapeptide -Asp.Ala.Ser.Val. The sequence of this peptide was established by subjecting the DNP peptide to partial acid hydrolysis. DNP-aspartic acid and DNP-aspartylalanine were isolated from the ether extract of the hydrolysate. The dipeptide DNP-serylvaline was isolated from the aqueous portion following a second treatment with DNFB. The presence of this tetrapeptide as the C-terminal grouping in ribonuclease is thus strongly inferred from the combined data above.

Partial Reconstruction of Ribonuclease Chain

Point I--Ribonuclease contains, as its N-terminal sequence, the grouping Lys.Glu.Thr.AIa- (1) and, as its C-terminal sequence, -Asp.Ala.Ser.Val.


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R. R. REDFIELD AND C. B. AYFINSEN 401

Point 2-Trypsin digestion of dinitrophenylated, oxidized ribonuclease leads to the production of five fragments labeled Peptides A, B, C, D, and E. These peptides contain the N-terminal and C-terminal amino acid residues and the amino acid groupings indicated in Fig. 5. The C-terminal sequence indicated for Peptide D is based on the isolation, described above, of the C-terminal arginine sequence from chymotrypsin digests of Peptide D.

Point %--In view of the end-group studies, the N-terminal lysine Peptide A is assignable to the N-terminal position of ribonuclease.

Point &Peptide E, since it lacks arginine, must be assigned to the C-terminal end of ribonuclease.

Point 5-The proper relative alignment of t,he five trypsin fragments below is dependent on knowledge of the structure of the arginine-containing sequences which form the ‘(bridges” between them. Three relatively small arginine-containing “bridges” have been isolated from acid and pepsin hydrolysates of ribonuclease. These “bridges” are (1) -Ser(Arg,Asp,

Pepfde Schematic Structure Lys.Glu.Thr.Ala...................................Glu.Arg

B Glu. . . . . . Arg

; Asp.Leu.Thr.Lys.Asp.Arg CyS08H. Ser.(Leu or lleu),Thr,CyS03H,Asp).Arg

E Glu........................................Asp.Ala.Ser.Val

FIG. 5. Partial structures of peptides produced during trypsin digestion of DNP-

oxidized rihonuclease.

Leu), (2) Asp.(Glu,Arg)-, and (3) -Glu.Arg.Glu.(Asp,Met,Ala,His,Thr, Ser& .

Point B-“Bridge” (3) contains methionine. The carboxyl group of the arginine residue in “bridge” (3) must join either Peptide B or E, since only these two are N-terminal glutamic acid peptides. Since, according to the analyses given earlier, Peptide E is devoid of methionine, arginine “bridge” (3) must constitute a juncture between Peptide B, on the right, with another of the major trypsin fragments on the left of arginine. Further, this latter peptide must have as its C-terminal sequence -Glu.Arg.

Point ~-AS shown in Fig. 5, neither Peptide C nor Peptide D obeys this restriction. It may therefore be concluded that Peptide B is preceded by Peptide A and we may write the tentative arrangement A-B-(D,C)-E.

Point 8--Arginine “bridge” (1) could have the following alternative structures: (1) .Ser.Rrg.Asp.Leu., (2) .Ser.Asp.Leu.Arg., (3) .Ser.Leu.Asp. Arg., or (4) .Ser.Asp.Arg.Leu. Structure (4) may be eliminated, since arginine must be followed by cysteic acid or by a dicarboxylic amino acid (Fig. 1). The arrangements indicated in structures (2) and (3), in which arginine is C-terminal, could occur only at the C-terminal end of Peptide


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B (Fig. 5). Since (Table I) Peptide B does not contain leucine, structures (2) and (3) are impossible. The sequence of arginine “bridge” (1) must thus be Ser.Arg.Asp.Leu, as indicated in structure (1).

The isolation of the arginine “bridge,” -Ser.Arg.Asp.Leu., together with the complete sequence of Peptide C, Asp.Leu.Thr.Lys.Asp.Arg, establishes the structure -Ser.Arg.Asp.Leu.Thr.Lys.Asp.Arg-, since the sequence, -Arg.Asp-, can occur only once in ribonuclease (only one N-terminal aspartic acid end-group is formed during trypsin digestion).

Point g--Therefore, Peptide C cannot follow Peptide D, since the C-terminal sequence of Peptide D (see Fig. 5) is incompatible with the arrangement of amino acids in Point 8. (There is no serine in juxtaposition to arginine in Peptide D.)

Point lo-Since Peptide C cannot follow Peptide D, it must precede it. The correct arrangement is, therefore, A-B-C-D-E.

Lys .Glu. Thr. Ala. (Ala2 , Lys)Phe . Glu . Arg . GZu . (Asp ,Thr , Sera ,Met ,His, Ala) . (Ala, Cys- ,Tyr , Seram4, Lys, Me&, Glu, Asps). Ser. Arg . Asp. Leu , Thr . Lys . Asp .Arg. Cys-. (Asp, Val, Pro ,Phe, (Thr , *Lys)) (Asp,, Glua, Sera , Thrz , Met, Gly , Ala,, Vald, His ,Lysr-2 ,Tyrt , C~s-2~~. Ser) . ((Leu or Ileu) ,Thr) . Cys- . Asp. Arg . Glu. (Gly , Ser) . (Asp,, Glut, Serr_z, Thr3, Ala2 , Prozml, Va13, Gly , (Leu -l- Ileu)z, Phe , Hisz, Lysz-3, Tyrs-) , Cys-2) . Asp. Ala. Ser. Val

FIG. 6. Partial reconstruction of the peptide chain of ribonuclease. *, as discussed in the text (Point 11 under “Partial reconstruction of ribonuclease chain”) Thr and Lys may also be included within this group of amino acids. This latter arrangement is compatible with the data of Hirs, Stein, and Moore (32).

Point II--The large peptide (Lys,Aspz,Arg,CyS03H,Val,Pro,Thr,Phe) described earlier, containing phenylalanine and arginine but no glutamic acid, may be included in the considerations as follows:

There are 3 phenylalanine residues in ribonuclease, 1 in each of Peptides A, D, and E (see Table I). The phenylalanine residues in Peptides A and E can only exist in a sequence together with arginine, if the sequence also includes glutamic acid (Fig. 5). The peptide (Lys,Asp,,Arg,CyS03H,Val,- Pro,Thr,Phe) must therefore be a part of Peptide D and must include the arginine “bridge” between Peptides C and D. This bridge may then be extended as follows: .(Thr,Lys,((-Asp.Arg.CySO~H-).(Val,Pro,Phe,~p))), where the position of the threonine and lysine residues cannot be fixed with respect to the two groups of residues in parentheses since these 2 residues could derive from either Peptide C or D.

Point 12-Glycine occurs only in Peptides D and E. In view of the nature of the arginine “bridge” between Peptides C and D (Point ll), which rules out the occurrence of glycine within 5 residues of arginine, the glycine-containing peptide (CySOsH,Asp,Arg,Glu,Gly,Ser) must occur at the bridge between Peptides D and E. This grouping may be further sub-


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divided, in view of the C-terminal sequence of Peptide D (Fig. 5) as follows: .(CyS03H,((.Asp.Arg.Glu).(Gly,Ser))).

Poin.t IS-On the basis of the points listed above, and the analyses listed in Table I, we may partially reconstruct the ribonuclease chain as shown in Fig. 6.

SUMMARY

The partially reconstructed formula for ribonuclease presented in this paper summarizes a large part of the results of our structure studies to date. The object of the present communication has also been to report on the feasibility and relative simplicity of the approach to structure determination which we have chosen,

The relatively incomplete structure given here already makes possible certain general observations concerning basic features of the enzyme. Most interesting, perhaps, is the amino acid composition of the C-terminal portion of the molecule which apparently contains 3 of the 4 (32) proline residues and 2 of the 4 histidine residues present in the protein. In view of the inactivation of ribonuclease brought about by photooxidation of histidine (33) and because of other studies (34, 35) suggesting that the active center of ribonuclease is located near the C-terminal end of the chain, these observations take on special interest in connection with further investigations nom in progress on the structural basis of the catalytic action of ribonuclease.

The approach to the elucidation of protein structure employed in these studies involves certain features which might become of particular advan- tage when larger proteins (having molecular weights of 30,000 or greater) are investigated. The specificity of trypsin, combined with the relative ease of isolation of arginine “bridges” for aligning trypsin fragments, makes it likely that any protein containing reasonable quantities of arginine may be susceptible to this general method. The present studies indicate that the separation of relatively large fragments, though rich in both dinitro- phenyl groups and in cysteic acid residues, offers no great difficulty when the techniques of electrophoresis in urea solutions and of chromatography in proper solvents are applied.

In concluding this preliminary report we would like to join Hirs, Stein, and Moore (32) in expressing our gratification over the valuable interchange of information which has taken place during the structural work by their group and ourselves. Although the present reconstruction of the ribonuclease molecule has purposely been made without reference to the Rocke- feller Institute contributions, it is important to observe that overlapping and consistent conclusions may be drawn from both sets of data. Thanks


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are due Dr. Fred Sanger for his generous hospitality and assistance to one of us (C. B. A.) during the summer of 1954 at the Department of Biochem- istry, University of Cambridge, Cambridge, England.

BIBLIOGRAPHY

1. Anfinsen, C. B., Redfield, R. R., Choate, W. L., Page, J., and Carroll, W. R., J.

Biol. Chem., 207, 201 (1954). 2. Him, C. H. W., Stein, W. H., and Moore, S., J. Biol. Chem., 211, 941 (1954). 3. Hirs, C. H. W., Moore, S., and Stein, W. H., J. Biol. Chem., 219, 623 (1956).

4. Sanger, F., Advances in Protein Chem., 7, 1 (1952). 5. Bergmann, M., and Fruton, J., Advances in Enzymol., 1, 63 (1941). 6. Green, N. M., and Neurath, H., in Neurath, H., and Bailey, K., The proteins,

New York, 2, pt. B (1954).

7. Sanger, F., and Tuppy, H., Biochem. J., 49, 481 (1951). 8. Li, C. H., Geschwind, I. I., Cole, R. D., Raacke, I. D., Harris, J. I., and Dixon,

J. S., Nature, 176, 687 (1955). 9. Bell, P. H., J. Am. Chem. Sot., 76, 5565 (1954).

10. White, W. F., and Landmann, W. A., J. Am. Chem. Sot., 77, 1711 (1955). 11. Hirs, Cl. H. W., Moore, S., and Stein, W. H., J. Biol. Chem., 200, 493 (1953).

12. Tanford, C., and Hauenstein, J. D., J. Am. Chem. Sot., in press. 13. Thompson, E. 0. P., Biochim. et biophys. acta, 16, 440 (1954). 14. Him, C. H. W., J. Biol. Chem., 219, 611 (1956).

15. Sanger, F., Biochem. J., 39, 507 (1945). 16. Northrop, J. H., and Kunitz, M., in Aberhalden, E., Handbuch der biologischen

Arbeitsmethoden, Berlin, Abt. IV, Teil 2, 2213 (1936). 17. Levy, A. L., Nature, 174, 126 (1954).

18. Blackburn, S., and Lowther, A. G., Biochem. J., 48, 126 (1951). 19. Sanger, F., Biochem. J., 46, 563 (1949). 20. Porter, R. R., in Gerard, R. W., Met.hods in medical research, Chicago, 3, 256

(1950). 21. Michl, H., Monatsh. Chem., 82, 489 (1951). 22. Redfield, R. R., and Anfinsen, C. B., Federation Proc., 14, 868 (1955).

23. Zahn, R., and Stahl, I., 2. physiol. Chem., 293, 1 (1953). 24. Redfield, R. R., and Barron, E. S. G., Arch. Biochem. and Biophys., 36,443 (1952). 25. Smith, E. L., and Stockell, A., J. Biol. Chem., 207, 501 (1954).

26. Redfield, R. R., Biochim. et biophys. acta, 10, 344 (1953). 27. Clayton, R. A., and Strong, F. M., Anal. Chem., 26, 1362 (1954). 28. Hirs, C. H. W., Stein, W. H., and Moore, S., Abstracts, American Chemical Soci-

ety, 126th meeting, New York, 89C, Sept. 12-17 (1954). 29. Anfinsen, C. B., Flavin, M., and Farnsworth, J., Biochim. et biophys. acta, 9, 468

(1952).

30. Ohno, K., J. Biochem., Japan, 40, 621 (1953). 31. Niu, I.-C., and Fraenkel-Conrat, H., J. Am. C’hem. Sot., 77, 5882 (lQ55). 32. Hirs, C. H. W., Stein, W. II., and Moore, S., J. Biol. Chem., 221, 151 (1956). 33. Weil, L., and Seibles, T. S., Arch. Biochem. and Biophys., 64, 368 (1955).

34. Anfinsen, C. B., Biochim. et biophys. acta, 17, 593 (1955). 35. Anfinsen, C. B., J. Biol. Chem., 221, 405 (1956).


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With the technical assistance of Juanita CookeRobert R. Redfield, Christian B. Anfinsen and

FRAGMENTSLARGE ENZYMATICALLY PRODUCED

AND RELATIVE ALIGNMENT OFII. THE PREPARATION, SEPARATION, THE STRUCTURE OF RIBONUCLEASE:

1956, 221:385-404.J. Biol. Chem.

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THE STRUCTURE OF RIBONUCLEASEshould obtain a mixture of fifteen peptides by complete trypsin digestion of oxidized ribonuclease, which contains 10 lysine and 4 arginine residues. This